Interferon beta-like molecules for treatment of stroke
The present invention relates to use of interferon beta-like polypeptides for treatment of stroke or transient ischemic attach in a primate, preferably in a human. More particularly, the interferon beta-like polypeptides differs from the amino acid sequence of wild-type human IFNB (SEQ ID NO:2) in that at least one glycosylation site, preferably at least one in vivo N-glycosyltaion site has been introduced. Optionally the interferon beta-like polypeptides are PEGylated.
1 .- 7 . (canceled)
8 . The method according to claim 39 or 44 , wherein said glycosylation site is an in vivo N-glycosylation site.
9 . The method according to claim 8 , wherein the IFNB variant is asialo-glycosylated.
10 . The method according to claim 39 or 44 , wherein the amino acid sequence of said variant differs from the amino acid sequence of wild-type human IFNB (SEQ ID NO:2) in 1-15 amino acid residues.
11 . The method according to claim 39 or 44 , wherein said at least one glycosylation site is introduced by a substitution selected from the group consisting of S2N+N4T/S, L9N+R11T/S, R11N, S12N+N14T/S, F15N+C17S/T, Q16N+Q18T/S, K19N+L21T/S, Q23N+H25T/S, G26N+L28T/S, R27N+E29T/S, L28N+Y30T/S, D39T/S, K45N+L47T/S, Q46N+Q48T/S, Q48N+F50T/S, Q49N+Q51T/S, Q51N+E53T/S, R71N+D73T/S, Q72N, D73N, S75N, S76N+G78T/S, L88T/S, Y92T/S, N93N+195T/S, L98T/S, E103N+K105T/S, E104N+L106T/S, E107N+E109T/S, K108N+D110T/S, D110N, F111N+R113T/S and L116N.
12 . The method according to claim 11 , wherein said substitutions are selected from the group consisting of S2N+N4T, L9N+R11T, Q49N+Q51T, R71N+D73T and F111N+R113T.
13 . The method according to claim 12 , wherein said substitutions are selected from the group consisting of 4Q9N+Q51T, R71N+D73T and F111N+R113T.
14 . The method according to claim 13 , wherein said substitutions are selected from the group consisting of Q49N+Q51T and F111N+R113T.
15 . The method according to claim 11 , wherein said variant comprises substitutions selected from the group consisting of
Q49N+Q51T+F111N+R113T,
Q49N+Q51T+R71N+D73T+F111N+R113T,
S2N+N4T+F111N+R113T,
S2N+N4T+Q49N+Q51T,
S2N+N4T+Q49N+Q51T+F111N+R113T,
S2N+N4T+L9N+R11T+Q49N+Q51T,
S2N+N4T+L9N+R11T+F111N+R113T,
S2N+N4T+L9N+R11T+Q49N+Q51T+F111N+R113T,
L9N+R11T+Q49N+Q51T,
L9N+R11T+Q49N+Q51T+F111N+R113T and
L9N+R11T+F111N+R113T.
16 . The method according to claim 15 , wherein said variant comprises the substitutions Q49N+Q51T+F111N+R113T.
17 . The method according to claim 39 or 44 , wherein the cysteine residue located at position 17 in human wild-type IFNB (SEQ ID NO:2) has been removed.
18 . The method according to claim 17 , wherein said cysteine residue has been removed by the substitution C17S.
19 . The method according to claim 18 , wherein said variant comprises substitutions selected from the group consisting of
C17S+Q49N+Q51T,
C17S+F111N+R113T,
C17S+Q49N+Q51T+F111N+R113T,
C17S+Q49N+Q51T+R71N+D73T+F111N+R113T,
S2N+N4T+C17S+F111N+R113T,
S2N+N4T+C17S+Q49N+Q51T,
S2N+N4T+C17S+Q49N+Q51T+F111N+R113T,
S2N+N4T+L9N+R11T+C17S+Q49N+Q51T,
S2N+N4T+L9N+R11T+C17S+F111N+R113T,
S2N+N4T+L9N+R11T+C17S+Q49N+Q51T+F111N+R113T,
L9N+R11T+C17S+Q49N+Q51T,
L9N+R11T+C17S+Q49N+Q51T+F111N+R113T and
L9N+R11T+C17S+F111N+R113T.
20 . The method according to claim 19 , wherein said variant comprises the substitutions C17S+Q49N+Q51T+F111N+R113T.
21 . The method according to claim 39 or 44 , wherein said variant comprises a substitution in position 110.
22 . The method according to claim 21 , wherein said substitution is selected from the group consisting of D110F, D110V, D110W and D110Y.
23 . The method according to claim 22 , wherein said substitution is D110F.
24 . The method according to claim 23 , wherein said variant comprises substitutions selected from the group consisting of
C17S+D110F+F111N+R113T,
C17S+Q49N+Q51T+D110F+F111N+R113T,
C17S+Q49N+Q51T+R71N+D73T+D110F+F111N+R113T,
S2N+N4T+C17S+D110F+F111N+R113T,
S2N+N4T+C17S+Q49N+Q51T+D110F+F111N+R113T,
S2N+N4T+L9N+R11T+C17S+D110F+F111N+R113T,
S2N+N4T+L9N+R11T+C17S+Q49N+Q51T+D110F+F111N+R113T,
L9N+R11T+C17S+Q49N+Q51T+D110F+F111N+R113T and
L9N+R11T+C17S+D110F+F111N+R113T.
25 . The method according to claim 24 , wherein said variant comprises the substitutions C17S+Q49N+Q51T+D110F+F111N+R113T.
26 . The method according to claim 25 , wherein said variant has the amino acid sequence shown in SEQ ID NO:3.
27 . The method according to claim 39 or 44 , wherein a polymer molecule is covalently attached to an amino acid residue of the variant, said amino acid residue comprising an attachment group for the polymer molecule.
28 . The method according to claim 27 , wherein said polymer is a PEG molecule.
29 . The method according to claim 27 , wherein said attachment group is the ε-amino group of a lysine residue or the N-terminal amino group.
30 . The method according to claim 27 , wherein at least one lysine residue has been removed.
31 . The method according to claim 30 , wherein said lysine residue is selected from the group consisting of K19, K33, K45, K52, K99, K105, K108, K115, K123, K134 and K136.
32 . The method according to claim 31 , wherein said lysine residue is selected from the group consisting of K19, K33, K45 and K123.
33 . The method according to claim 30 , wherein said lysine residue has been removed by substituting said lysine residue with an arginine or glutamine residue.
34 . The method according to claim 33 , wherein said substitution(s) is (are) selected from the group consisting of K19R, K33R, K45R, K123R, K19R+K33R, K19R+K45R, K19R+K123R, K33R+K45R, K33R+K123R, K45R+K123R, K19R+K45R+K123R, K19R+K33R+K123R, K19R+K33R+K45R, K33R+K45R+K123R and K19R+K33R+K45R+K123R.
35 . The method according to claim 34 , wherein said substitutions are selected from the group consisting of K19R+K45R+K123R, K19R+K33R+K123R, K19R+K33R+K45R and K33R+K45R+K123R.
36 . The method according to claim 35 , wherein said substitutions are selected from the group consisting of K19R+K33R+K45R.
37 . The method according to claim 36 , wherein said variant comprises substitutions selected from the group consisting of
C17S+Q49N+Q51T+K19R+K33R+K45R,
C17S+D110F+F111N+R113T+K19R+K33R+K45R,
C17S+Q49N+Q51T+D110F+F111N+R113T+K19R+K33R+K45R,
C17S+Q49N+Q51T+R71N+D73T+D110F+F111N+R113T+K19R+K33R+K45R,
S2N+N4T+C17S+D1110F+F111N+R113T+K19R+K33R+K45R,
S2N+N4T+C17S+Q49N+Q51T+D110F+F111N+R113T+K19R+K33R+K45R,
S2N+N4T+L9N+R11T+C17S+D110F+F111N+R113T+K19R+K33R+K45R,
S2N+N4T+L9N+R11T+C17S+Q49N+Q51T+D110F+F111N+R113T+K19R+K33R+K45R,
L9N+R11T+C17S+Q49N+Q51T+D110F+F111N+R113T+K19R+K33R+K45R and
L9N+R11T+C17S+D110F+F111N+R113T+K19R+K33R+K45R.
38 . The method according to claim 37 , wherein said variant comprises the substitutions C17S+Q49N+Q51T+D110F+F111N+R113T+K19R+K33R+K45R.
39 . A method for treating or preventing stroke or cerebrovascular accident (CVA) in a primate, the method comprising administering an effective amount of an interferon β (IFNB) polypeptide variant comprising an amino acid sequence which differs from the amino acid sequence of wild-type human IFNB (SEQ ID NO:2) in that at least one glycosylation site has been introduced, to a primate in need thereof.
40 . The method according to claim 39 , wherein said stroke is ischemic stroke.
41 . The method according to claim 40 , wherein said ischemic stroke is selected from the group consisting of embolic stroke, cardioembolic stroke, thrombotic stroke, large vessel thrombosis, lacunar infarction, artery-artery stroke and cryptogenic stroke.
42 . The method according to claim 39 , wherein said stroke is hemorrhagic stroke.
43 . The method according to claim 42 , wherein said hemorrhagic stroke is selected from the group consisting of intraparenchymal stroke, subdural stroke, epidural stroke and subarachnoid stroke.
44 . A method for treating or preventing transient ischemic attack in a primate, the method comprising administering an effective amount of an interferon β (IFNB) polypeptide variant comprising an amino acid sequence which differs from the amino acid sequence of wild-type human IFNB (SEQ ID NO:2) in that at least one glycosylation site has been introduced, to a primate in need thereof.
45 . The method according to claim 39 or 44 , wherein said primate is a human.
46 . (canceled)